pRB is one of the best-known tumor suppressors. It plays a central role in the processes that enable cells to stop dividing and to permanently withdraw from the cell cycle. Consistent with its role as a key negative regulator of cell proliferation, pRB's ability to function is thought to be compromised in most, if not all, human cancer cells. The majority of human tumors do not mutate the RB1 gene but acquire changes, such as mutations in the locus encoding the p16 cdk inhibitor that reduce pRB's activity. Despite extensive investigation, the molecular details of pRB's mechanism of action are vague. More than 150 different proteins have been reported to physically interact with pRB and have been proposed to be relevant for pRB function. However, most studies of have investigated just pRB-associated protein, or a small group of pRB-interactors, at any one time. Different groups have championed the importance of individual interactions but because the literature on pRB-associated proteins has accumulated in a piecemeal fashion, there is no information about the relative significance of these interactions, and no consensus over which pRB- associated proteins are genuinely important. We propose to take advantage of the rapid developments in shRNA technologies to systematically test the effect of knocking-down each of the reported pRB-associated protein on a panel of pRB-induced phenotypes. This will enable us to assess the relative importance of each pRB-binding protein for various activities of pRB. To identify pathways that impinge on these protein/protein interactions we will complement this analysis by screening the same set of functional assays with a collection of shRNAs that target the kinome. Preliminary data shows that these function-based screens not only identify proteins that are needed for pRB to act, but also identify pathways that can be targeted to enhance specific outcomes, including the ability of pRB to induce senescence in human tumor cells. Using pancreatic cancer cells as an example of cancers that retain an intact RB gene, we will identify the interacting proteins that are essential for pRB-mediated suppression of cell proliferation, and we will test whether targeting co-operative pathways can enhance the activity of endogenous pRB and improve its ability to induce a permanent cell cycle arrest. Together, these experiments will provide a framework for future analysis of pRB function and will uncover ways to enhance the activity of endogenous pRB in human cancer cells.